A novel mutation approach, namely, the atmospheric and room temperature plasma, was used to treat the spores of Aspergillus oryzae shanghai brewing 3.042 for the selection of high activity of acid protease producer. 30 mutants with high acid protease activity was firstly screened according to the different appearances of colonies and the rate of transparent zone contrast to colony diameter, calculating the activity of acid protease, a mutant B2 producing high-output, stable protease was obtained. The acid protease activity of strain B2 increased 45.8% compared with shanghai brewing 3.042, and its hereditary stability is invariant after 10 generation.
Key words: ARTP; mutation and screening; acid protease; Aspergillus oryzae
1 Introduction
Atmospheric and Room Temperature Plasma (ARTP) is a newly technology for mutation and screening of microbial genome. Because of its low voltage radio-frequency power source, low temperature plasma, equal jet flow of plasma, great diversity of mutation library, easy operation and high security, ARTP is popular with microbial breeding workers increasingly.
Aspergillus oryzae plays an important role in the fermentation of soy sauce, and is widely used in the manufacture of traditional fermented food. It grows rapidly, has a high ability to produce spores and secrete various and vast amounts of hydrolytic enzymes. In addition to the characteristic have said, Aspergillus oryzae is normally GRAS (generally regard as safe) strains because of the absence of aflatoxin gene. At present, Aspergillus oryzae shanghai brewing 3.042 is adopted for the fermented soy sauce industry in our country generally. Its extracellular enzyme system is composed of protease, amylase, glucoamylase, cellulase, phytase and other enzymes, however, the acid protease is not sufficient for the hydrolysis of proteins in wheat and soybean.
In soy sauce fermentation culture, koji making determined the utilization of raw proteins and the quality of soy sauce. During the koji making, microorganism, such as mold, yeast and other bacterium can secrete large number of exoenzyme and other organics. It has been proven that the activity of neutral and alkaline protease is the major protease compared to acid protease while the value of pH in environment drop to acid at the middle and later stage of fermentation. So raw material is not been utilized sufficiently.
It has important significance to breeding the strain with high acid protease activity for the soy sauce fermentation industry. Although the genetic engineering technology make great achievements in breeding with the advance of molecular genetics, its security is still been questioned because of the introducing of foreign genes. In this paper, the ARTP mutation system is employed in the mutation of Aspergillus oryzae shanghai brewing 3.042 and a new strain with high acid protease activity is obtained.
2 Materials and Methods
2.1 Bacterial strain and medium
The original strain is Aspergillus oryzae shanghai brewing 3.042 (from College of Bioengineering, Tianjin University of Science and Technology ).
Potato dextrose agar (PDA) medium: peeled potato, 200 g; add 1000 mL water and boiled 30 min; filter by gauze; dextrose, 20 g; agar, 20 g; sterilized at 121℃ for 15 min ).
Screening medium: nonfat-dried milk, 1%; agar 1.5%; sterilized at 115℃ for 20 min respectively; mixed at sterile condition and adjust the pH value to 3.0.
Fermentation medium: bean pulp: bran: water =6: 4: 8; sterilized at 121℃ for 30 min.
2.2 Reagent and instrument
Casein and Folin-ciocalteu's phenol reagent, company of Sigma; the rests are analytical reagents; bran and bean pulp are purchased in the market.
ARTP mutation system, Si Tsingyuan (BEST) Biotechnology; TU-1810 ultraviolet and visible spectrophotometer, Purkinje General; optical microscope, OLYMPUS; TCL-12 high speed freezing centrifuge, Institute of Biophysics of Chinese Academy of Sciences; BS124S analytical balance, Sartorius Scientific Instruments(Beijing) Co., Limited;
2.3 Mutation method
2.3.1 Spores suspension preparation
Aspergillus oryzae spores were washed from a 3-day agar slant culture with 10 mL sterile normal saline, and inoculated in 250 mL conical flask which contains 10 glass bead, then incubated at 30℃.
Spores were dispersed and activated after 1 h and then were filtrated by sterile filter paper. The spores suspension was diluted to 106 cells/mL.
2.3.2 ARTP mutation
Iron dish preparation: 10 μL spores suspension was spreaded over iron dish under aseptic condition, then was dry by airing.
Mutation conditions: radio-frequency voltage was 120W, gas flow was 10 L/min, the distance between ion source and iron dish was 2 mm, the optimal exposure time was 140 s.
2.4 Breeding method
2.4.1 Prescreen process
After the iron dish had been exposed for optimal time, the samples were placed in a sterile Ep tube and eluted with sterile normal saline for the mutated spore solution. 100 μL mutated spore suspension was spread on the solid screening medium and cultivated at 37℃ for 2-3 days. Then the colonies with high rate of transparent zone contrast to colony diameter or different morphologies were isolated.
Isolates were activated by inoculate on PDA solid medium for 2-3 days. The activated strains were pick by toothpick and inoculated on solid screening medium. Four strains were cultured on every plate.
2.4.2 Rescreening procress
Inoculum development: spores were eluted from PDA medium and diluted to 106 cells/mL.
Fermentation: 2 mL spores suspension was added to a 500 mL conical flask with 35 g fermentation medium and cultured at 30℃ for 48 h, shake the flasks every 6 h to make the koji dispersed.
Crude enzyme preparation: 5 g fermented medium was mixed with lactic acid buffer (pH=3.0) and shaking for 1 h at 40℃, then the mixture was centrifuged at 12,000 rpm for 10 min at 4℃. The acid protease will be in the supernatant.
Detection: the activity of acid protease was determined by the Folin-phenol Reagent Method. Proteolytic activity was that one unit of enzyme activity was defined as the amount of enzyme that produce 1 μg tyrosine per min under assay conditions. 1 mL preheated crude enzyme extract and 1 mL casein (1%, soluted by lactic acid buffer, pH=3.0 ) were mixed. The reaction system was incubated at 40℃ for 20 min and reaction was arrested by adding 2 mL of 0.4 mol·L-1 trichloroacetic acid (TCA). The reaction mixture was placed at room temperature for 10 min and then centrifuged at 12,000 rpm for 5 min at 4℃. 1 mL supernatant was collected and added to a test tube with 5 mL of 0.4 mol·L-1 Na2CO3 and 1 mL of diluted Folin-phenol reagent. Then the tube was incubated for 20 min at 40℃ and the absorbance was read at 660 nm.
3 Results and Discussion
3.1 Lethality rate of Aspergillus oryzae by the ARTP
3.1.1 Determination of the parameter of ARTP for mutation
According to the previous study, the variable operating parameter for ARTP main include radio frequency power, gas flow rate, the distance between plasma generator and sample plate, the kind of plasma working gas and the treatment time. In this study, treatment time was designed as the variable quantity and other conditions were displayed in Table 1.
Table 1 Operating conditions using ARTP
Parameters
Operating condition
Power input（W）
Treatment distance（mm）
Gas flow rate （L/min）
Treatment time（s）
Sample amount
Gas
115
2
10
0, 30, 60, 90……210, 240
10uL
Helium
3.1.2 The variation of strains after treatment by ARTP
3.1.3 Survival rate of Aspergillus oryzae treated by ARTP mutation
Treatment time was the unique factor after the above-mentioned parameters were determined. Figure 1 shows the survival rate of Aspergillus oryzae treated by ARTP with respect to treatment time.
Fig.1 Variation of the survival rate of the A.oryzae with the ARTP treatment time.
The survival rate of the spores under different treatment time was calculated in accordance with the following equation:
Survival rate = (S/U)Ã-100%
where S is the total CUF of mutant strains, and the U is the total CUF of the sample without treatment.
Figure 3 shows the survival rate of spores of A.oryzae treated by the ARTP with respect to treatment time. Through calculating the survival curve, it was found that the survival curve according with the "saddle" curve which has good correlation with the curve caused by ion implantation. It can be discovered that the survival rate of treated spores decreased to 30.1%, 10.0% and 7.5%, respectively, after treated with the ARTP for 120, 150 and 180 s. When the spores was treated for 240 s under same condition, nearly no spores could survive. Therefore, in this article, the optimal exposure time was 140 s to obtain an efficient survival rate.
Other studies have shown that microbial mutation breeding process is a combined impact with DNA damage and DNA repair. In the long process of evolution, microbes have developed a serious of DNA damage repair mechanisms in order to maintain the genetic stability, so DNA damage was not always lead to the change of phenotype.
3.2 Equation of enzyme activity
Fig.2 Determination of Abs-Tyr standard curve
The relationship between absorbance and the concentration of L-Tyr was shown in Figure 2. Hence, the formula for acid protease activity was as follow:
Enzyme activity = Ã-4Ã-N
where A is the weight of L-Tyr (μg) according to the absorbance, 4 stands for 1 mL reacting solution was take from 4 mL, and N is the dilution rate of crude enzyme solution.
3.3 Screening procedure
The isolation and screening of mutants was one of the keys in microbial breeding, besides the optimal mutation condition. To screen more productive protease-secreting microorganisms efficiently, the adopted method had to follow the principles: (a) the transparent zone should be tremendous compared to original strain; (b) the scale of bacterial colony should be massive enough to guarantee the biomass in fermentation progress. The detection of acid protease making microorganisms was based on the theory that acid protease catalyze the opaque milk protein medium to transparent zone in acid environment. Because of the large samples in breeding stage, screening procedure was consisted of preliminary screening and re-screening.
3.3.1 Preliminary screening
In the preliminary screening period, the morphology of colony and the rate of transparent zone contrast to colony diameter were the dominating factors. The morphology of mutants may change to be diversity because of the genome mutation, usually. However, the variation of appearance did not means the improvement of acid protease activity but the transformation of gene sequence. Previous studies has revealed that the phenotype of organisms was the common formation of genotype and environment.
The existing models in mutants screening is still depend upon the transparent zone. After mutant spores were spread on the solid screening medium and cultivated for 3 days, the value of transparent zone contrast to colony diameter was calculated in Table 2.
Table 2 rate of transparent zone contrast to colony diameter
Strains
3.042
A-1
A-3
A-5
A-7
A-8
A-9
A-14
A-18
B-1
Transparent zone（mm）
16.7
15.4
13.5
16.1
15.2
14.6
13.9
14.3
15.2
16.1
Colony（mm）
13.4
11.2
10.7
12.48
12.0
11.7
11.0
10.9
11.8
12.4
Ratio
1.25
1.37
1.26
1.29
1.27
1.25
1.26
1.31
1.29
1.30
Strains
B-2
B-3
B-6
B-8
B-11
B-13
B-19
B-20
B-22
B-25
Transparentzone（mm）
17.2
13.5
17.5
16.6
14.3
14.2
14.6
17.2
16.4
13.2
Colony（mm）
12.6
10.4
13.8
13.0
11.0
11.3
11.6
13.5
13.1
10.1
Ratio
1.37
1.30
1.27
1.28
1.30
1.26
1.26
1.27
1.25
1.30
3.3.2 Re-screening
As above-mentioned, prescreening do not provide enough evidence to determined the activity of acid enzyme, it just present us with a rough instruction. In order to obtain a fine strain possessed of practical application value, the re-screening procedure is necessary to pick out the strain with high-activity. The acid protease activity of above-mentioned 20 strains were assayed by section 2.4.2. Enzyme activity was showed in the Table 3.
Table 3 acid protease activity of mutant strains
Strains
3.042
A-1
A-3
A-5
A-7
A-8
A-9
A-14
A-18
B-1
U（U/g）
75.4
119.2
78.2
65.4
81.5
76.3
87.4
90.1
73.3
90.7
Strains
B-2
B-3
B-6
B-8
B-11
B-13
B-19
B-20
B-22
B-25
U（U/g）
105.5
85.3
70.0
98.3
76.1
95.3
78.5
87.4
89.6
79.3
Compared Table 2 and Table 3, a phenomenon can be obtained that there is not a liner relation between the enzyme activity and the rate of transparent zone to colony diameter. In solid fermentation procedure, the acid protease activity is related to not merely the ability of secreting enzyme but also the biomass in koji making process.
According to the detection method, the enzyme activity was the performance of all protease in acid environment. At the acid condition, acid protease plays a leading role in catalyze protein, whereas it doesn't means the incompetence of other protease in the protein katabolism. So the acid protease activity in this paper doesn't speak very strictly.
3.4 The genetic stability of mutants
In mutation breeding industry, the genetic stability is vital for the fermentation stage. After treatment by physical, chemical, or biological mutagens, the yield of protease may be improved at the earlier generation because of the back mutation. Others' research revealed that gene repair and proofreading occurred in the mutant gene is one of the main reason for the spawn degeneration. The usual method to avoid unstable protease-overproducing is to breed strains, then assay the activity of protease.
Among the examined strains, five mutants, A-1, B-1, B-2, B-8 and B-13, had higher enzyme activity than that of the wild strain by 58.1%, 20.3%, 39.9%, 30.4% and 26.4%, respectively. After 10-generation culture, the mutant B-2 still maintained high acid protease activity. Enzyme activity of mutants after ten generation was showed in Figure 3.
Fig.3 Enzyme activity of mutants after ten generation
The strain B-2 was isolated to lucubrate its stability in fermentation progress. Acid protease activity of B-2 in front 10 generation were presented in Figure 4.
Fig.4 Increase rate of B2's acid protease activity
3.5 Total enzyme activity of B-2
According to the research have accomplished above, a high activity strain of B-2 was isolated and increased with average 45.8%. Its genetic stability also meets the soy sauce production requirements in industry very well. A.oryzae was the main protease secreting microorganism in koji making, and A.oryzae shanghai brewing 3.042 was more famous for its high activity of neutral and alkaline protease in fermentation industry. Therefore, the B-2 should possessed of high acid protease activity at the same time high neutral and alkaline protease activity can be used for the soy sauce producing.
Fig.5 Enzyme activity of 3.042 and B-2
Figure 5 presents the acidã€neutral and alkalinity protease activity of strain B-2 and shanghai brewing 3.042. Compared to original strain 3.042, the mutant strain B-2 obtained a increase rate of 45.8% and 16.7% of acid and neutral protease activity, respectively, while the alkaline protease has a decrease rate of 10%.
4 Conclusions
The study demonstrated that ARTP mutant system was an efficient method for the selection of improved strains of acid protease, and has a high mutation rate. It has been proven that it is the chemically active species that break the double chains of DNA, instead of UV, intense electric field, heat and charged particlesã€å‚考】. The effect of plasma on microorganism depend on the operating conditions, such as power input, treatment distance, gas flow rate and the treatment time.
With the continuous development of mutation breeding, the method for artificial mutation breeding is become popular increasingly. In this paper, a mutant Aspergillus oryzae strain, namely B-2, was obtained and have a huge potential for industry application. The B-2 mutant exhibited a high acid protease activity compared to the original strain. The acid and neutral protease activity increased by 45.8% and 16.7%, respectively. However, the alkaline protease have a decline of 10%. After comprehensive study of 3.042 and B-2, we discovered that B-2 is superior to 3.042 in utilization of raw materials, amino acid productivity and even the flavor of soy sauce.
